The overall goal of this project is to discover novel chemical probes that are capable of potently and selectively disrupting the CXCL12/CXCR4 protein-protein binding. The chemokine CXCL12 and its G protein-coupled receptor CXCR4 are high-priority clinical targets because of their involvement in metastatic cancers (also implicated in autoimmune disease and cardiovascular disease). A number of chemokines have been validated as targets for drug development, but virtually all discovery efforts focus on the agents that bind GPCRs. However, disrupting the extensive chemokine- receptor interface by CXCR4 receptor antagonists has proven difficult due to unmanageable toxicities for traditional drug discovery approaches, emphasizing the need for alternative strategies focusing on agents that bind the CXCL12 chemokine. Recently, we developed a novel structure-based hybrid in silico/NMR screening strategy and identified a CXCL12 ligand 2 that occludes the receptor recognition site. We optimized the initial hit 2 by designing and synthesizing a small fragment library containing only nine tetrazole derivatives. From this library, we discovered two fragments with higher ligand efficiency (LE) than the typical PPI disruptors (LE d 0.24). One fragment 5 exhibited a Kd of 13 M (LE=0.33) for CXCL12 binding in sY12 site and one fragment 9 with a Kd of 41 M (LE=0.28) in sY21 site measured by 2D NMR. We also solved an X-ray co-crystal structure of CXCL12 with tetrazole fragment 9 at 1.8 resolution in sY21 site. Based on these findings, we designed and synthesized a second library containing a dozen compounds. We have demonstrated that the nature of the CXCL12 binding site is conductive to obtaining higher affinity ligands that exhibit reasonable activity in functional chemotaxis assays. The best compound 9c, from a total of ~20 compounds synthesized in two round of optimization, showed an IC50 of 51 M in CXCL12-induced chemotaxis, a 16 fold improvement from the original hit 2. We propose to develop these validated CXCL12 chemokine antagonists into in vivo chemical probes for use in diagnosis and treatment of metastatic disease. The aims of this revised grant application are: 1) Design, synthesize and optimize CXCL12 chemokine antagonists targeting the sY21 binding site on CXCL12. 2) Design, synthesize and optimize CXCL12 antagonists in sY12 and sY7 binding sites. 3) Design, synthesize high-affinity inhibitors using fragment-growing and linking strategy. 4) Develop potent and selective inhibitors as in vivo chemical probes using robust bioassays of calcium flux, chemotaxis and migration in vitro and metastasis in colon and melanoma mouse models as well as structural determination of co-complexes using X-ray and NMR. Because chemokine signaling plays a key role in cancer cell migration, CXCL12 chemokine antagonists are likely to find utility as novel therapeutic agents for metastatic disease. Finally, we will assess the in vivo efficacy of optimized compounds using animal models for CXCR4-directed metastatic cancer as described in our recent publication in PNAS. This is a key prerequisite for PAR-12-060.